Process for the isolation of certain physiologically-active substances



picric, picrolonio 100 mg. of insulin hydrochloride of very 55N.CsH4.NOa small activity were dissolved in 50 cc. of water Hop l| I andprecipitated with a solution of barium mud-Jinn, picrate. Theprecipitate formed was cenfl vi i trifuged 01f, stirred up] with waterand OH centrifuged as long as the aqueous extract con- 60 which areinsoluble or only slightly soluble. vicinity of 0.1 mg. per clinicalunit. 7

' 2 fication. The salts which have been used grof the posterior lobewere secured. The 75 40 above named acids, without taking the excesswith a solution of neutral lead acetate. After so Patented Feb; 4, 1930i UNITED" STATES PATENT OFFICE CASIMIR FUNK, OF NEW YORK, N. Y.

PROCESS FOR THE ISOLATION OF C I lRTAIN PHYSIOLOGICALLY-ACTIVESUBSTANCES N0 Drawing. Application filed January 4, 1927, Serial No.159,018, and in Germany January '15, 1926.

Whereas it is known that variousfsub- (antiscorbutic). The process usedmay be 'starices form picrates which, however, rarely best described bythe following examples.

separate out on account of their solubility, it

has now been found that by using salts of Ewamp Pmflmtion of insulintinued to' contain some soluble material; in Hols 1 this way the-bulk ofimpurities was eliminated in form of water soluble barium salts.

The yield of the purified insulin amounted to I 55 mg. and whereas theactivity of the crude 5 I I product could not be ascertained on account(naphthol-yellow S) and other analogous of irregularity of action onrabbits (in nitro-,nitrooxy and nitro and nitrooxy sulfovicinity of 0.45mg. per clinical unit), the acids characteristic double salts are formedactivity of the purified material was in the It has been found that mostof the substances 4 tested form combinations usually with one EwamplePmpamtwn of the hormone of salt and not with another, a behavior whichthe posterior of the pztmtfly 9W greatly assists their separation andidenti- From 270 pituitary glands from the ox 60 chiefly are the saltsof copper, barium, lead, material was extracted with diluted alcohol.silver and mercury. The procedure is usually After evaporation 0 the S vn n l carried o t as follo tion of the lipin material the aqueousextract 1 Aqueousor alcoholic extracts (the latter was precipitated witha solution of silver being evaporated and taken up with water) picrate.The precipitate amounted. to ca. 80 of various biological materials,such as glands 150 mg. and yielded after elimination of silver of,internal secretion, tissue extracts or exand picric acid a crystallinematerial, which tracts containing water soluble vitamines. arerepresented the totality of the active material precipitated as a rule,first with a saturated of the posterior portion of the gland.

solution of icric acid but only in those cases 85 in which iicric acidalone does not carry Pfepammmwf mmmmesBM/ZD downany of the activematerial. The filtrate fl kgr. of rice polishings were extracted of thepicric acid precipitation is then suc with water and the aqueous extractafter cessively treated with the various salts of the evaporation waspurified by precipitation of the salts out of the solution, and the theelimination'of lead the extract was confractions obtained are tested bythe known centrated in vacuum and precipitated with biological andpharmacological methods. picric acid. The filtrate which contained Thenew method has been applied, among the totality of the active materialwas treated other, to the purification of insulin, to the first withbarium picrate, which was found 05 preparation of the active substancesof posnot to carry down any of the active mateterior lobe of thepituitary, to the preparation rial, and then either with silver picrate,or of the active substances of the ovary and for with mercury picratefiit with both salts comthe preparation of the vitamines B(antiberibined. On decomposing latter salts almost beri), D (yeastgrowth promoting) and C the total amount of vitamine B can be ob- 1 wasevaporated in vacuum and the residue tained. Thus it has been found thatsilver picrate precipitates about 60% of vitamine B, mercuric picrate80%, silver'flavianate- 70%, mercury flavianate90%, whereas the silverpicrolonate only 20% and barium and lead picrate none. Most of thevitamine I) was found in the mercury picrate precipitate. Same resultswere obtained with yeast.

Emample 4. The preparation of the amtiscorb'utio m'tamz'ne (oitamine 0)From 45 lemons the juice which amounted to about 1 liter hasbeenextracted and the juice precipitated first with a solution ofneutral lead acetate and then the active material with basic leadacetate. After the decomposition of the latter lead salt the filtratewas precipitated with silver picrate. The

silver salt which amounted to about 1 gr..

yielded on decomposition the total vitamine C present in this fraction.

It will thus be seen that the present invention comprises the use ofsalts of salt-forming aromatic nitro com ounds in the separation andpurification of physiologically and pharmacologically active biologicalmaterials, such as hormones and vita-mines; that salts of aromat1cnltro-phenols, nitro-sulfonic ac1ds and nitrophenol-sulfonlc ac1ds areof particular value; and that the heavy metal saltsvof these aromaticnitro compounds are of particular interest.

I claim:

1. In the separation and purification of physiologically andpharmacologically active biological materials the use of salts ofsaltforming aromatic nitro compounds as selective precipitants, of-saidmaterials and asso-- ciated impurities.

2. In the separation and purification of hormones and vitamines, the useof heavy metal salts of salt-forming aromatic nitro compounds asselective precipitants of said hormones and vitamines and associatedimpurities.

3-. In the separation and purification of water soluble vitamines theuse of heavy.

metal salts of salt forming aromatic nitro compounds as selectiveprecipitants of said vitamines and associated impurities. I

4. In the separation and purification of antiscorbutic vita-mine, theuse of heavy metal salts of salt-forming aromatic nitro compounds asselective precipitant of said vita- 6. In the separation andpurification of physiologically and pharmacologically active 1ologicalmaterials the use of salts of saltforming aromatic nitro-hydroxycompounds as selective recipitants of said materials and associatedimpurities.

In testimony whereof, I aflix my signature.

CASIMIR FUNK.

